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The aquaponic system is an alternative strategy to treat aquaculture waste and achieve food independence. Bacteria play vital roles in the aquaponic system as they can transform ammonia or ammonium into nitrite and then into nitrate, which is more favorable for bacteria, fish, and plants. The objective of this study was to determine the effect of nitrifying bacteria (Nitrosomonas europaea Winogradsky and Nitrobacter winogradskyi Winslow) on the aquaponic system in terms of water quality, nutrient availability, and productivity of carp (Cyprinus carpio), lettuce (Lactuca sativa var. crispa), and vetiver grass (Chrysopogon zizanioides L.). The experiment consisted of four treatments: aquaculture of carp as a control for fish (A), hydroponic of lettuce and vetiver grass without nutrient addition as a control for plants (B), aquaponic (carp, lettuce, vetiver grass) (C), and aquaponic with nitrifying bacteria addition (D). The results showed nitrifying bacteria addition had a significant effect on daily growth rate (DGR) and relative growth rate (RGR) of lettuce within a treatment; on the other hand, the nitrifying bacteria did not give a significant effect to RGR of vetiver grass. The growth rate, specific growth rate, and survival rate of the carp in aquaculture treatment (A) were lower than in both aquaponic treatments (C and D). Nitrifying bacteria addition in the aquaponics system had a significant effect of increasing the orthophosphate concentration. Water quality was also indicated to be better in the aquaponic system than in the aquaculture system. The integration of aquaculture and hydroponics with the addition of nitrifying bacteria enables the formation of microorganism communities, nitrate, and orthophosphate, which lead to the improvement of water quality, nutrient availability, and plant growth.

The CO2 fixation by autotrophic microbes has an important significance for improving carbon sequestration in composting. In this work, the succession of autotrophic CO2-fixing bacterial communities was investigated using quantitative PCR and high-throughput sequencing of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes (cbbL and cbbM). The results presented that the number of autotrophic CO2-fixing bacteria was comparable to that in the soil, and most species have not been classified into known bacterial groups, only part of them was assigned into Proteobacteria and Actinobacteria. Phylogenetic analysis displayed that the dominant known cbbL-containing bacteria were Allochromatium vinosum, Rhodobacter sphaeroides, Nitrobacter winogradskyi, Paracoccus yeei and Porphyrobacter sp. CACIAM 03H1, while the dominant known cbbM-containing bacteria were Sulfuritalea hydrogenivorans, Pseudomonas resinovorans, Achromobacter xylosoxidans, Sphingopyxis macrogoltabida and Thermomonospora curvata. In addition, canonical correspondence analysis showed that the evolution of autotrophic CO2-fixing bacterial communities was greatly affected by physico-chemical parameters such as temperature, C/N and pH.

Several regions of the world suffer from water quality degradation caused mainly by anthropogenic interference, domestic sewage discharge, industrial discharges, and contaminants from agricultural activities, which increase the amount of nutrients into water such as nitrogen and phosphorus intensifying the process of eutrophication. Considered as one of the main problems faced by supply water sources in the world, eutrophication represents a global phenomenon that also affects the socioeconomic and environmental sector. In the present study, a bacterial pool formed by the Nitrosomonas europae, Nitrobacter winogradskyi, and Paracoccus denitrificans strains was used to remove the compounds NH3, NO2−, NO3−, P, and PO43−, real water samples from human supply at time 0 to 24 h. At the final time (24 h), the bacterial pool obtained total removal (100%) of all compounds analyzed. With the removal of nutrients from water, there was also reduction in the amount cyanobacteria and saxitoxins of water. Thus, the bacterial pool can be applied as a remediators of micropollutants such as compounds N and P, since their performance in removel the compounds was satisfactory. The removal of micropollutants from water bodies aims to decrease the nutrients responsible for cyanobacteria blooms, eutrophication, as well as improve water quality, optimize, and reduce water treatment costs, benefiting both the aquatic ecosystem and human health.

Background: The aim of this study was the enrichment of high-performance microbial communities in biofilters for removal of ammonium and nitrite from aquaculture water. Methods: Ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were enriched from different environmental water samples. The microbial communities with higher ammonium and nitrite removal activity were selected and adapted to different temperatures [9 ºC, 15 ºC, room temperature (25 ºC), and 30 ºC]. The expression of genes involved in nitrification including ammonia monooxygenase (AMO) and nitrite oxidoreductase (NXR) were measured in temperature-adapted AOB and NOB microbiomes. The microbial species present in the selected microbiomes were identified via 16s rRNA sequencing. Results: The microbial communities containing Nitrosomonas oligotropha and Nitrobacter winogradskyi showed the highest ammonium and nitrite removal activity at all temperatures used for adaptation. Furthermore, the microbial communities do not contain any pathogenic bacteria. They also exhibited the highest expression of AMO and NXR genes. Using the enriched microbial communities, we achieved a 288% and 181% improvement in ammonium and nitrite removal over the commonly used communities in biofilters at 9 °C, respectively. Conclusions: These results suggest that the selected microbiomes allowed for a significant improvement of water quality in a recirculating aquaculture system (RAS).

The objective of this study was to evaluate the effects of salt accumulation on nitrifying bacterial communities in a nitrifying bioreactor combined with forward osmosis. The conversion of nitrite to nitrate was inhibited at a total dissolved solids (TDS) concentration of 17.3 g/L, whereas conversion of ammonia to nitrite was inhibited at a higher concentration (52.8 g-TDS/L). The gene copies of ammonia-oxidizing bacteria (AOB) were more abundant than those of nitrite-oxidizing bacteria (NOB) throughout the entire operating period of 225 days. Among NOB, the number of copies of Nitrobacter spp. were 100–1000 times higher than those of Nitrospira spp. A total of 140 operational taxonomic units were identified using 454 pyrosequencing. The relative abundances of autotrophic AOB and NOB accounted for 34.1–57.8% during 225 days. Dominance of Nitrosomonas eutropha was stable as a salt-tolerant AOB, but the representative NOB, Nitrobacter winogradskyi, showed salt-sensitive variations in their relative abundance. Nonmetric multidimensional scaling and hierarchical clustering analysis clearly illustrated the shift in bacterial community due to external conditions, i.e., ammonia loading rate, alkalinity availability, and salinity. Heterotrophic bacteria contributed to changes in overall bacterial community structure in the nitrifying osmotic membrane bioreactor despite the absence of carbon sources in the influent.

For many years, the generation of nitrates from organic sources in order to create nutrient solutions for hydroponics had proved a challenge until lately when microorganisms were introduced to perform this task. The objectives of the current study therefore were to use local microbial consortium to nitrify goat manure in water and to determine microbial diversity in the inoculated consortium. Therefore, microorganisms were sourced from garden soil and natural compost at the Sam Nujoma Marine resources Research Centre (SANUMARC) in Henties Bay Namibia to convert organic nitrogen in goat manure from Utuseb farm near Walvis Bay into nitrates. Results show that microbial consortium from the compost source produced significantly (P< 0.05) more nitrates followed by the garden-soil source, suggesting that it is necessary to add inoculum in order to generate nitrate from goat manure. The ammonia oxidising bacteria (AOB) community from the compost sample’s was dominated by uncultured ammonia-oxidising species followed by uncultured bacterium (both not identified), with the least being Nitrosomonas species. The AOB community from the garden source was dominated by uncultured bacterium, followed by uncultured ammonia-oxidising species and the least being Nitrosomonas species. NOB community from the compost source was dominated by uncultured bacterium, followed by Nitrobacter winogradskyi and Nitobacter vulgaris with the least being Nitrococcus mobilis and Nitrospira moscovensis. For the garden soil microbial source, uncultured nitrite-oxidising bacteria dominated followed by uncultured bacterium, whereas the least species were N. moscovensis and Nitrobacter alkalicus. Moreover, community composition of the compost sample was more diverse than the community from the garden sample. These results maintain that there are other unculturable yet important microbes doing the same job if not better than the known ones, in this case suggesting that there may be other local nitrite-oxidizing bacteria responsible for oxidizing ammonia other than the traditionally known Nitrobacter, Nitrospira and Nitrococcus species. Key words: Organic hydroponics, ammonification, nitrification, ammonia-oxidizing bacteria, nitrite-oxidizing bacteria.

Nitrobacter is an important chemolithoautotrophic nitrite-oxidizing bacteria and has exhibited potential for the oxidation of nitrite to nitrate in natural and engineered ecosystems. Microbial kinetics of Nitrobacter have been widely reported and are used to model the nitrite oxidation process and optimize the design parameters of the biological processes for wastewater treatment. However, limited information is available about the nitrite oxidation kinetics of the two coexisting Nitrobacter stains, namely the Nitrobacter winogradskyi and the Nitrobacter hamburgensis. The current work mainly focuses on the influence of temperature on the kinetic characteristics of the coexistence of N. winogradskyi and hamburgensis, which were enriched in a lab-scale sequencing batch reactor (SBR) through 419 operating cycles. The results for the kinetic properties of nitrite oxidation of the enrichments within the temperature range of 15–35 °C showed that the maximum specific rate (rmax), half-saturation coefficients (KS), growth-rate constants (μmax), and temperature coefficient (θ) lied within the ranges of 22.91–93.58 mM/g/d, 0.13–2.47 mM, 0.026–0.105 1/d, and 1.038–1.137, respectively. The maximum activity and nitrite affinities of tested microorganisms were sensitive to temperature. Furthermore, the relationships between temperature and normalized kinetic parameters (rmax, μmax, and θ) were developed as: 0.069T − 0.076 (15–30 °C), 0.0057T − 0.062 (15–30 °C), and 5.7 T 40.9 + T + T 2 10.5 (15–35 °C), respectively. Moreover, the capabilities of N. winogradskyi and N. hamburgensis to oxidize nitrite were lost at the temperature of 35 °C and were non-recoverable at temperature of 25 °C. Overall, the findings of the present work can provide available data for future study about the mathematic models of nitrification kinetics and ecological niche differentiation of Nitrobacter species.

Despite increasing interests in osmotic membrane bioreactors, the information regarding the bacterial toxicity effects of reversely transported draw solute (RTDS) is limited. In this study, two representative draw solutes (NaCl and MgCl2) were used at different concentrations (0, 2.5, 5.0, 7.5 and 10.0 g/L) to evaluate their toxicity in a continuous nitrifying bioreactor. Notably, Mg2+ selectively inhibited the activity of ammonia-oxidizing bacteria (AOB), which decreased to 11.3% at 7.5 g-Mg2+/L. The rRNA-based analysis was more effective than the rDNA-based analysis to elucidate the relationship between active communities of nitrifying bacteria and the actual nitrifying performance. Nitrosomonas europaea, a representative AOB, was vulnerable to Mg2+ in comparison to Na+. In contrast, the dominant nitrite-oxidizing bacteria (NOB), Nitrobacter winogradskyi and Nitrolancea hollandica, maintained a relevant level of relative abundance for achieving nitrite oxidation after exposure to 10 g/L Na+ and Mg2+. This fundamental inhibition information of the draw solute can be applied to set the operational regime preventing the critical solute concentration in mixed liquor of nitrifying OMBRs.

An integrated photo-bioelectrochemical system (IPB) for wastewater treatment combines a microbial fuel cell with an algal bioreactor, eliminating requirements for aeration, promoting electricity generation, remediating nutrients and producing algal biomass for conversion into biofuel or other bioproducts. To examine strategies for improving IPB functions of electrochemical output and nutrient removal efficiency, this study tested effects of cathode bacterial inoculation and nitrogen loading on cathode microbial community and IPB performance. IPB cathodes were inoculated with the green alga Chlorella vulgaris, in combination with nitrite-oxidizing bacteria (NOB) Nitrobacter winogradskyi, and/or ammonium-oxidizing bacteria (AOB) Nitrosomonas europaea. IPB performance was examined before and after nitrifying bacteria inoculations and under three ammonium loading concentrations in the wastewater medium. Bacterial communities in the cathode suspension and biofilm were examined by 16S rRNA gene sequence analysis. Relative to the algae only control, cathode inoculation with NOB and/or AOB improved net nutrient removal, but resulted in reduced dissolved oxygen availability, which impaired electricity generation. Higher ammonium loading increased electricity production and nutrient removal, possibly by overcoming algal-bacterial competition. Inoculation with nitrifying bacteria resulted in minor changes to total bacterial composition and AOB or NOB comprised3% of total sequences after 1 month. Community composition changed more dramatically following increase in ammonium-N concentration from 40 to 80 mg L

The effects of water permeation intensity on nitrifying bacterial activity, forward osmosis (FO) membrane performance, and bacterial community structure were investigated in an osmotic membrane bioreactor (OMBR). Ammonia was partially oxidized by controlling acidification buffer concentration. Then, the FO module was externally integrated with a nitrifying bioreactor to examine nitrifying bacterial activity in response to the increased salinity. The salinity gradually increased from 2.7 to 45.7 g.L−1 total dissolved solids (TDS). Inhibition of nitrite-oxidizing activity was initiated at a salt concentration of 36.2 g.L−1 TDS. Complete ammonia-oxidizing bacteria inhibition occurred at 45.7 g.L−1 TDS. Illumina high-throughput sequencing revealed that the bacterial community responded to the operational conditions such as the ammonia loading rate and salt concentration. Only two species, Nitrosomonas eutropha and Nitrobacter winogradskyi, were responsible for nitrification in the OMBR. The cellulose triacetate FO membrane showed poor rejection efficiencies for ammonia, nitrite, and nitrate. The FO module was capable to maximally recover 107.8% water in the OMBR. However, the water permeation intensity should be appropriately adjusted to avoid nitrification failure by maintaining the optimal salinity as lower than 35 g.L−1 TDS, which is equivalent to water permeation efficiency of 107.8% in this study.

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH3) to nitrate (NO3−) via nitrite (NO2−), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO2− oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l- Histidine effectively restored cell growth and NO2− oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation.

Typically, nitrification is a two-stage microbial process and is key in wastewater treatment and nutrient recovery from waste streams. Changes in salinity represent a major stress factor that can trigger response mechanisms, impacting the activity and the physiology of bacteria. Despite its pivotal biotechnological role, little information is available on the specific response of nitrifying bacteria to varying levels of salinity. In this study, synthetic communities of ammonia-oxidizing bacteria (AOB Nitrosomonas europaea and/or Nitrosomonas ureae) and nitrite-oxidizing bacteria (NOB Nitrobacter winogradskyi and/or Nitrobacter vulgaris) were tested at 5, 10, and 30 mS cm-1 by adding sodium chloride to the mineral medium (0, 40, and 200 mM NaCl, respectively). Ammonia oxidation activity was less affected by salinity than nitrite oxidation. AOB, on their own or in combination with NOB, showed no significant difference in the ammonia oxidation rate among the three conditions. However, N. winogradskyi improved the absolute ammonia oxidation rate of both N. europaea and N. ureae. N. winogradskyis nitrite oxidation rate decreased to 42% residual activity upon exposure to 30 mS cm-1, also showing a similar behavior when tested with Nitrosomonas spp. The nitrite oxidation rate of N. vulgaris, as a single species, was not affected when adding sodium chloride up to 30 mS cm-1, however, its activity was completely inhibited when combined with Nitrosomonas spp. in the presence of ammonium/ammonia. The proteomic analysis of a co-culture of N. europaea and N. winogradskyi revealed the production of osmolytes, regulation of cell permeability and an oxidative stress response in N. europaea and an oxidative stress response in N. winogradskyi, as a result of increasing the salt concentration from 5 to 30 mS cm-1. A specific metabolic response observed in N. europaea suggests the role of carbon metabolism in the production of reducing power, possibly to meet the energy demands of the stress response mechanisms, induced by high salinity. For the first time, metabolic modifications and response mechanisms caused by the exposure to salinity were described, serving as a tool toward controllability and predictability of nitrifying systems exposed to salt fluctuations.

Herein, we report on composite materials of biologically active microorganisms placed in a synthetic polymer matrix. These so-called "living composites" were utilized for gold sequestration (Micrococcus luteus) and bioremediation of nitrite (Nitrobacter winogradskyi) to demonstrate functionality. For the preparation of the living composites the bacteria were first encased in a water-soluble polymer fiber (poly(vinyl alcohol), PVA) followed by coating the fibers with a shell of hydrophobic poly(p-xylylene) (PPX) by chemical vapor deposition (CVD). The combination of bacteria with polymer materials assured the stability and biologically activity of the bacteria in an aqueous environment for several weeks.

Biocomposites, i.e., materials consisting of metabolically active microorganisms embedded in a synthetic extracellular matrix, may find applications as highly specific catalysts in bioproduction and bioremediation. 3D constructs based on fibrous biocomposites, so-called "artificial biofilms," are of particular interest in this context. The inability to produce biocomposite fibers of sufficient mechanical strength for processing into bioactive fabrics has so far hindered progress in the area. Herein a method is proposed for the direct wet spinning of microfibers suitable for weaving and knitting. Metabolically active bacteria (either Shewanella oneidensis or Nitrobacter winogradskyi (N. winogradskyi)) are embedded in these fibers, using poly(vinyl alcohol) as matrix. The produced microfibers have a partially crystalline structure and are stable in water without further treatment, such as coating. In a first application, their potential for nitrite removal (N. winogradskyi) is demonstrated, a typical challenge in potable water treatment.

Hygienic fecal treatment in resource-oriented sanitation (ROS) systems is an important concern. Although the addition of nitrifying microorganisms is a sustainable fecal treatment method in ROS systems, it is essential to examine the cleanliness of this method. In this study, we investigated the fate of fecal indicators in source-separated fecal samples through tracking Escherichia coli and total coliforms. The effects of adding different amounts of Nitrosomonas europaea bio-seed, along with a constant amount of Nitrobacter winogradskyi bio-seed, were studied. In intact feces samples, the pathogen population underwent an initial increase, followed by a slight decrease, and eventually became constant. Although the addition of nitrifying microorganisms initially enhanced the pathogen growth rate, it caused the reduction process to become more efficient in the long-term. In addition to a constant concentration of 10,000 cells of N. winogradskyi per 1 g feces, a minimum amount of 3000 and 7000 cells of N. europaea per 1 g feces could completely remove E. coli and total coliforms, respectively, in less than 25 days. Increasing the amount of bio-seeds added can further reduce the time required for total pathogen removal.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2(-)) as a source of energy and nitrogen. In most natural situations, NO2(-) oxidation is coupled closely to ammonium (NH4(+)) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4(+) to meet its N-biosynthetic requirements. Interestingly, NH4(+) delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4(+) while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4(+) concentration. Genes in assimilatory NO2(-) metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4(+). Nitrobacter winogradskyi grown in medium with 25 mM NH4(+) upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4(+) and is likely to be modified during coupled nitrification with NH3 oxidizers.

The aim of this study was to investigate ammonia load removable by a fixed-bed bioreactor. In this study, high ammonia loads, up to 4.5 kgN/m3 d were applied on a fixed bed reactor colonised by two pures nitrifying bacteria: Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391. The loads were similar to the highest values reported in literature especially with a two bacteria population. The complete ammonia removal efficiency in our installation has reached 2.5 kgN/m3.d for a full nitrification. An N-tank in series model was used to represent the fixed bed reactor and to predict the nitrification in the installation. The developed model was accurate and representative of the nitrification assumed by Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391. This model was also used to predict the biomass and oxygen concentrations during the experiment under various operational conditions. The biofilm fixed on the solid support (polysterene beads 2 mm diameter) was mainly present in the bottom of the reactor where the ammonia load was applied. It is demonstrated that in this region of the column the oxygen concentrations reached the lowest values.

Resource-oriented sanitation (ROS) systems play an important role in handling source-separated human sanitary wastes intended to be used for other purposes. Usually, the purpose of employing such systems is to render the source-separated human feces suitable as fertilizer or soil conditioner. However, the high volume, low degradation rate, and lack of fertility management pose challenges to such enterprises. Accordingly, treatment by applying controlled amounts of nitrifying microorganisms could be useful. The effect of adding different amounts of Nitrosomonas Europaea bio-seed, along with a certain amount of Nitrobacter Winogradskyi bio-seed, to source-separated human feces was investigated. The results show that adding 7000-8000 or more N. Europaea cells, along with 10,000 N. Winogradskyi cells, to 1 g feces, resulted in up to 90% degradation of the organic matter by enhancing the growth of heterotrophic microorganisms. Moreover, the nitrogen composition and pH of the degraded feces were optimized to meet the criteria for standard fertilizer. The results can be useful for managing source-separated feces in ROS systems in accordance with the specific aims of such systems, i.e., reducing feces volume by bio-degradation and increasing the fertility to meet the standard criteria for fertilizer.

Nitrite has a key role in nitrogen conversion during composting. In this study, the dynamic changes in the NO2- contents, abundances of nirS and nxrA, and the bacteria that harbored these genes were determined during composting. NO2- accumulated during the initial composting stage. The nirS gene was abundant throughout composting, whereas the nxrA gene was only abundant in the late composting phases. Ralstonia sp. and Thauera sp. were the dominant denitrifiers that harbored nirS, and Nitrobacter winogradskyi Nb-255 was the dominant nitrifier that harbored nxrA. Structural equation modeling showed that NO2- was mainly reduced by nirS in the early phases, and oxidized by nxrA in the late phases, but especially in the maturity phase. Network analysis showed that the dominant bacteria harboring nirS and nxrA were hubs in the modules related to the reduction and oxidation of NO2-, and they had competitive relationships during the cooling and maturity phases.

The biological filter capable of simultaneous nitrification and denitrification was constructed for aquatic animal experiments in the International Space Station (ISS). The biological filter will be used to remove harmful ammonia excreted from aquatic animals in a closed water circulation system (Aquatic Habitat). The biological filter is a cylindrical tank packed with porous glass beads for nitrification and dual plastic bags for denitrification. The porous beads are supporting media for Nitrosomonas europaea and Nitrobacter winogradskyi. The N. europaea cells and N. winogradskyi cells on the porous beads, oxidize the excreted ammonia to nitrate via nitrite. On the other hand, the dual bag is composed of an outer non-woven fabric bag and an inner non-porous polyethylene film bag. The outer bag is supporting media for Paracoccus pantotrophus. The inner bag, in which 99.5% ethanol is packed, releases the ethanol slowly, since ethanol can permeate through the non-porous polyethylene film. The P. pantotrophus cells on the outer bag reduce the produced nitrate to nitrogen gas by using the released ethanol as an electron donor for denitrification. The biological filter constructed in this study consequently removed the ammonia without accumulating nitrate. Most of the excess ethanol was consumed and did not affect the nitrification activity of the N. europaea cells and N. winogradskyi cells severely. In accordance with the aquatic animal experiments in the ISS, small freshwater fish had been bred in the closed water circulation system equipped with the biological filter for 90 days. Ammonia concentration daily excreted from fish is assumed to be 1.7 mg-N/L in the recirculation water. Under such conditions, the harmful ammonia and nitrite concentrations were kept below 0.1 mg-N/L in the recirculation water. Nitrate and total organic carbon concentrations in the recirculation water were kept below 5 mg-N/L and 3 mg-C/L, respectively. All breeding fish were alive and ate the feed well. The results show that the nitrification and denitrification abilities of the biological filter sufficed to keep water quality for aquatic animal experiments in the ISS. This simple and effective system is certainly applicable to aquarium systems and aquaculture systems.

During long-term extra-terrestrial missions, food is limited and waste is generated. By recycling valuable nutrients from this waste via regenerative life support systems, food can be produced in space. Astronauts' urine can, for instance, be nitrified by micro-organisms into a liquid nitrate fertilizer for plant growth in space. Due to stringent conditions in space, microbial communities need to be be defined (gnotobiotic); therefore, synthetic rather than mixed microbial communities are preferred. For urine nitrification, synthetic communities face challenges, such as from salinity, ureolysis, and organics. In this study, a synthetic microbial community containing an AOB (Nitrosomonas europaea), NOB (Nitrobacter winogradskyi), and three ureolytic heterotrophs (Pseudomonas fluorescens, Acidovorax delafieldii, and Delftia acidovorans) was compiled and evaluated for these challenges. In reactor 1, salt adaptation of the ammonium-fed AOB and NOB co-culture was possible up to 45mScm-1, which resembled undiluted nitrified urine, while maintaining a 44±10mgNH4+-NL-1d-1 removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15±6mg NO3--NL-1d-1 production rate for 1% and 10% synthetic and fresh real urine, respectively. Batch activity tests with this community using fresh real urine even reached 29±3mgNL-1d-1. Organics removal in the reactor (69±15%) should be optimized to generate a nitrate fertilizer for future space applications. ispartof: SYSTEMATIC AND APPLIED MICROBIOLOGY vol:42 issue:6 ispartof: location:Germany status: published

Respirometry, which consists in the measurement of the oxygen uptake rate, is a method commonly used to determine kinetic and stoichiometric parameters of biological processes. In the present work, a novel microrespironetry method, which involves standard respirometry procedures but in a microreactor array, was developed and assessed, based on a standard commercial apparatus. First, the volumetric oxygen transfer coefficient ( K L a ), which is important for the interpretation of respirometric data, was determined under an exhaustive set of experimental conditions. K L a ranged from 2.2 to 48.0 h −1 , which is similar to K L a typically found in standard respirometers. Then, the method was assessed through the determination of the maximum specific growth rate, the half-saturation constant and the growth yield of axenic and mixed cultures of autotrophic and heterotrophic bacteria. The selected cultures were Pseudomonas putida F1, Nitrobacter winogradskyi , an activated sludge and a nitrifying consortium. Additionally to reporting parameters for these cultures, this report shows that the microrespirometry method allowed for the determination of stoichiometric and kinetic parameters. Simultaneous tests done with a single culture sample, made possible by the microreactor array, allowed parameters determination with a lower standard error than observed with traditional respirometry and with much less experimental effort.

The aim of this study was to obtain a nitrite-oxidizing bacterium with high nitrite oxidation activity for controlling nitrite levels. A nitrite-oxidizing bacterium, ZS-1, was isolated from the water of a coastal Pseudosciaena crocea-rearing pond. The strain was identified as Nitrobacter winogradskyi based on the phylogenetic analyses of the 16S ribosomal ribonucleic acid gene and nxrA sequence of ZS-1. Under aerobic condition, the nitrite-oxidizing activity of ZS-1 did not change considerably in the range of pH 7–9, but was strongly inhibited by lower (pH = 6) and higher (pH = 10) pH values. The optimum temperature range is 25–32 °C. Lower temperature made the adaptive phase of ZS-1 longer but did not affect its maximum nitrite oxidization rate. The nitrite-oxidizing activity of ZS-1 started to be inhibited by ammonia and nitrate when the concentrations of ammonia and nitrate reached 25 mg L−1 and 100 mg L−1, respectively. The inhibition was stronger with higher concentration of ammonia or nitrate. The nitrite-oxidizing activity of ZS-1, however, was not inhibited by high concentration of nitrite (500 mg L−1). The nitrite-oxidizing activity of ZS-1 was increased by low ammonia concentration (1 mg L−1 to 10 mg L−1).

Immobilized Spirulina mat was co-cultured with Pacific white shrimp (Penaeus vannamei) postlarvae for water quality control in a closed re-circulating system. During the culture period, the experimental Spirulina mat system showed total ammonia nitrogen and nitrate accumulation rates lower by 32.92 and 32.26 %, respectively, compared with the system without the mat. The water exchange rate in the system with the mat was comparatively reduced by 40 %. Moreover, survival rate, growth rate, and size (length) of the co-cultured shrimp larvae were greater than those of the control (P

Presence of different forms of ammonia in the wastewater treatment plants are toxic to aquatic life and promote eutrophication in receiving water bodies. It becomes imperative to lower the ammonia concentration in the effluents for which characterization of microbial diversity is foremost objective. Study was planned to identify the diversity of nitrifiers and denitrifiers from activated sludge using PCR technique. Cultural techniques revealed high bacterial diversity was found in the aeration tank where majority of the isolates belongs to class γ proteobacteria followed by β proteobacteria suggesting the adaptability of these groups. Presence of Nitrosomonas europaea revealed better ammonium oxidation condition at longer SRT of 6 and 7 d with DO concentration of 2.5 mg/L. As a part of NOB, Nitrospira sp. found to be more dominant than Nitrobacter winogradskyi as its detection was achieved in all samples of 6 and 7 d SRT, while later was detected only on 7 d SRT. Use of cultural techniques in combination with PCR analysis with 16S rRNA approach revealed Pseudomonas aeruginosa as important denitrifier. Highest removal efficiencies of COD, BOD, TN and NH 4 + – N were found to be 81, 78, 70 and 69% respectively at optimized SRT of 7 d.

The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: μ max = 0.054 h −1 , decay rate b = 0.003 h −1 and maintenance rate m = 0.135 gN-NH 4 + ·gX −1 ·h −1 for Nitrosomonas europaea ; μ max = 0.024 h −1 , b = 0.001 h −1 and m = 0.467 gN-NO 2 − ·gX −1 ·h −1 for Nitrobacter winogradskyi . A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.

Quorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO2, and N2O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi. We added a recombinant AiiA lactonase to N. winogradskyi cultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirK and nirK cluster genes (ncgABC) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi. Production and consumption of NOx increased and production of N2O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable., IMPORTANCE Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi, a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO2, and N2O) in N. winogradskyi. This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming.

Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi , in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi , several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism. IMPORTANCE Aerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and their neighboring microbial community.

Wastewaters from swine farms, nitrogen-dealing industries or side-stream processes of a wastewater treatment plant (e.g., anaerobic digesters, sludge thickening processes, etc.) are characterized by low C/N ratios and not easily treatable. In this study, a hollow fiber-membrane biofilm reactors (HF-MBfR) system consisting of an O2-based HF-MBfR and an H2-based HF-MBfR was applied for treating high-strength wastewater. The reactors were continuously operated with low supply of O2 and H2 and without any supply of organic carbon for 250 d. Gradual increase of ammonium and nitrate concentration in the influent showed stable and high nitrogen removal efficiency, and the maximum ammonium and nitrate removal rates were 0.48 kg NH4(+)-N m(-3) d(-1) and 0.55 kg NO3(-)-N m(-3) d(-1), respectively. The analysis of the microbial communities using pyrosequencing analysis indicated that Nitrosospira multiformis, ammonium-oxidizing bacteria, and Nitrobacter winogradskyi and Nitrobacter vulgaris, nitrite-oxidizing bacteria were highly enriched in the O2-based HF-MBfR. In the H2-based HF-MBfR, hydrogenotrophic denitrifying bacteria belonging to the family of Thiobacillus and Comamonadaceae were initially dominant, but were replaced to heterotrophic denitrifiers belonging to Rhodocyclaceae and Rhodobacteraceae utilizing by-products induced from autotrophic denitrifying bacteria. The pyrosequencing analysis of microbial communities indicates that the autotrophic HF-MBfRs system well developed autotrophic nitrifying and denitrifying bacteria within a relatively short period to accomplish almost complete nitrogen removal.

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO 2 − to Cl − and O 2 . Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira . Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase ( K m = 90 μM; k cat = 190 s −1 ; k cat / K m = 2.1 × 10 6 M −1 s −1 ), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.

Nucleic acid methods based on sequencing of clone libraries provide sequence and the phylogenetic information of an individual clone. In the present study, ammonia monooxygenase (amoA), nitrite-oxido-reductase(norB), nitrite reductase (nirS) and nitrous oxide reductase (nosZ) genes are chosen to detect nitrifiers and denitrifiers in coastal aquaculture using gene-specific primers. The abundance of these functional genes revealed the presence of nitrifying and denitrifying organisms in coastal aquaculture. Nitrifying and denitrifying communities were analyzed by parallel DNA extractions and clone library construction for amoA, norB, nirS and nosZ obtained from coastal aquaculture soil. Amino acids in parts of the amoA, norB, nirS and nosZ encoded proteins were aligned to know conserved amino acid residues. The amoA genes exhibited 81–82% identity to Nitrosomonas europaea, Nitrosococcus mobilis and Nitrosomonas eutropha which were also similar to particulate methane monooxygenase (pmoA) gene sequences. The norB genes are closely affiliated with Nitrobacter winogradskyi and other uncultured beta-proteobacteria. The nosZ genes exhibited 78–82% identity with Marinobacter sp. and other uncultured denitrifying gamma-proteobacteria. The present study could be useful for making bioremediation strategy for nitrogenous metabolites and understanding of nitrogen fluxes generated through these two functional groups in coastal shrimp aquaculture.

The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ∼21 kb of a ∼28-kb “autotrophic” island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b 561 , and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter “subcore” genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris . Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter .